Oxalate is excreted in the urine via a combination of both glomerular filtration and proximal tubular secretion. The intestinal absorption of oxalate may in turn be a major determinant of its excretion in the urine and the natural history of stone disease. We and others have shown that the apical anion exchanger Slc26a6, known to be expressed in the murine proximal tubule and intestine, is capable of mediating Cl'-oxalate, SO42" -oxalate, Cl'-formate, CI'-HCCy, and CI'-OH" exchange. Direct measurement of oxalate transport in Xenopus oocytes has revealed that several other SLC26 and SLC4 exchangers are capable of oxalate transport, indicating that there is significant molecular heterogeneity of epithelial oxalate exchange. We propose in Aim 1 to systematically evaluate the role of SLC26 exchangers and SLC4 exchangers in intestinal oxalate transport, so as to define the major transporters responsible for transepithelial oxalate absorption by this tissue. The kinetics, pharmacology, and electrophysiology of oxalate transport will thus be evaluated and characterized by heterologous expression of individual cDNAs in Xenopus oocytes. We will also characterize oxalate transport mechanisms in the Caco-2 intestinal epithelial cell line, in which we will assess the quantitative role of specific exchangers using RNA interference. Within the kidney, the apical membrane of the proximal tubule contains mechanisms capable of Cl'-oxalate, oxalate-base, and SO42"-oxalate exchange;Slc26a6 is clearly capable of all these activities and is likely the dominant apical oxalate exchanger in these cells. Apical oxalate exchange mediated by Slc26a6 is thus thought to function in proximal tubular secretion of oxalate, in concert with basolateral oxalate exchange mediated by Slc26a1. To clarify the role of Slc26a6 in the proximal tubule we will generate mice in Aim 2 with loxP sites flanking exons 5-7 of the Slc26a6 gene ("floxed mice"), for cell type-specific deletion of these exons. These mice and/or mice with a germline deletion of exons 5-7 will initially be utilized to examine the contribution of the Slc26a6 protein to apical oxalate exchange in the proximal tubule, using a variety of techniques. Finally, a significant fraction of patients with calcium-oxalate stones exhibit hyperabsorption of ingested oxalate, such that genetic variation in the major intestinal oxalate transporters identified in Aim 1 may predispose to nephrolithiasis. Using the resources and expertise of the Genetics Core we will define the spectrum of genetic variability in specific SLC26 and SLC4 genes, beginning with the apical exchanger SLC26A6. The functional consequence of non-conservative coding sequence variation in the SLC26A6 gene will thus be studied using heterologous expression in Xenopus oocytes. We.will also determine the functional consequences of coding sequence variation in WNK4, a novel kinase with potent inhibitory effects on both SLC26A2 and SLC26A6, apical oxalate exchangers in the small intestine. This phenotypic characterization will be correlated with the impact of these polymorphisms on the genetic predisposition to stone disease and hyperoxaluria, as determined in Project 3.